PCR Procedure
If you do not have pre-made plates, see Making PCR plates. #The DNA extracted by BIO can be found in the fridge on the second floor, beside the incubator. #Use pre-made frozen PCR plates (taken from the freezer on the second floor of BIO in the lysis room, beside the freezer where Pro K is kept), or you can make your own (instructions on the next page). #If you have access to a heat sealer, turn it on now to heat up. #Centrifuge the DNA plate and the PCR plate at 1000 g for 1 minute, or until they are no longer frozen. Centrifuge the PCR plates for about 10-20 sec. #Label the PCR plate with the primer, the name of the box, PCR, the date, and your name. #The PCR plate has clear self-adhering plastic to seal it. To remove the plastic carefully, start at a corner and peel it off on an angle (be careful since the benches are slippery and they could slide off, be sure to keep one hand on the plate to hold it). #The DNA plate will be covered by self-adhering foil, which has a tendency to rip while it is being removed. If it is to difficult to remove, use a clean tip to pry at the foil. #Make sure that the DNA plate is in the same alignment as the PCR plate. #Using a 10 μl multi-channel pipette, transfer 2 μl of DNA from the DNA plate to the PCR plate (do NOT put PCR mix into the DNA plate because this will contaminate your DNA with primers). #Look at the tips to make sure that each one has sucked up fluid from the DNA plate. If one didn’t work, push the tip on and try it again. Touch the tips to the bottom of the PCR plate and push down to the hard stop, remove the pipette from the wells and then release. Discard the tips. #Use new tips for every row as not to contaminate anything. #Seal the DNA plate with self-adhering foil. If there is a heat sealer, put the plate on the appropriate place and put the heat-sealing paper on it silver side down, white side up. Push down until it touches the top for 3 seconds. Push as hard as you can for about 15 seconds. Let go. Use a roller to seal the plate. Be really careful around the corners and the sides because sometimes the samples evaporate. #If there isn’t a heat sealer, just use the cap strips and make sure they are on tight. #Put the DNA plate in the freezer. #Centrifuge the PCR plate for about 10 seconds at 1000g. Be sure that the centrifuge is properly balanced. #Place the plate in the thermocycler and put the plastic mat on top of the plate and pull the cover over the plate to close it. On the Thermocycler the password is 12345, then: ::*▼ to get to hanner. ::*Press Enter ::*▼ to get to: ::::::*COIFAST for mini or full lep primers, Folmer primers ::::::*Fish52 for fish primers ::::::*MamCOI54 for mammal primers ::*Press Start at the bottom of the screen. ::*It will say Tubes, you have to press Enter and then ▲to get to Plate. ::*Press Enter. ::*Press OK at the bottom of the screen. :17. After about two hours your reaction will be complete, and the green light will turn red. Press Enter to unlock the machine. To program the thermocycler see Programing the Thermocycler. Make sure to put your PCR Plates into LIMS. The next step is to do the E-gel PCR Product Check. Return to Barcoding in the Hanner Lab Wiki. Updated April 27 2009